This invention relates to detection of pre-eclampsia. In particular, it relates to early diagnosis of pre-eclampsia by detecting elevated levels of the hormone inhibin A.
Pre-eclampsia, otherwise known as gestational proteinuric hypertension (GPH) is a multisystem disease of pregnancy of unknown cause. The maternal syndrome is characterised by various abnormalities: increased blood pressure, oedema, proteinuria and abnormal clotting, liver and renal function. The cause of pre-eclampsia is believed to lie in the placenta and there is evidence for a circulating endothelial cell xe2x80x9ctoxicxe2x80x9d factor which is most likely to originate from the syncytiotrophoblast. The syncytiotrophoblast provides the surface of contact between the placenta and the maternal blood and is a multi-nucleated syncytium with an extensive microvillous brush border.
Pre-eclampsia is a relatively common condition, occuring in approximately one in ten pregnant women. Around 10% of cases will be severe. In severe cases, early delivery may be necessary and there is therefore the risk of the child being handicapped. Pre-eclampsia also remains a danger to the lives of both babies and mothers.
The symptoms of pre-eclampsia described above are generally detectable from around 28 weeks up to full term and are not normally apparent before 24 weeks. The conventional tests are for kidney failure by measuring urea in the blood or protein in the urine, and for increased maternal blood pressure. There are some other indicators of pre-eclampsia, including hormonal ones. However, none of these tests can predict the on-set of pre-eclampsia more than a few days in advance of secondary symptoms becoming fully apparent.
If pre-eclampsia could be detected earlier, before clinical symptoms arise, it would be possible to intervene in affected pregnancies e.g. by treating with anti-hypersensitive drugs, increasing monitoring and foetal surveillance and thereby improve foetal and maternal outcome.
Thus, there is a need for a new test for pre-eclampsia and in particular a test that can predict the on-set of the secondary symptoms of pre-eclampsia earlier than existing tests. Other tests are routinely carried out during pregnancy e.g. spina bifida is tested for at 16 weeks. If pre-eclampsia could be tested for several weeks in advance of the possible onset of symptoms, more time would be available for treatment to avoid the possibility of serious problems arising.
It has now been discovered that the hormone inhibin A is significantly increased in cases of pre-eclampsia compared to normal pregnancies. Before secondary symptoms of pre-eclampsia are detectable, a group of patients who went on to develop pre-eclampsia showed a higher mean inhibin A level than a second group who did not go on to develop pre-eclampsia.
Inhibin A is a member of the family of inhibins which are heterodimeric proteins consisting of xcex1xcex2A (inhibin A) and xcex1xcex2B (inhibin B) subunits. The term xe2x80x9cinhibin Axe2x80x9d as used herein refers to the dimeric protein, which is the biologically active form of inhibin A. The two protein subunits are joined together by disulphide bonds. Inhibin A is produced mainly by the ovaries and has an endocrine role in inhibiting pituitary follicle stimulating hormone (FSH) production. In pregnancy, circulating levels of inhibin A are increased with the placenta being the major source (Muttukrishna et al 1995). In contrast, inhibin B levels are not elevated in either control or pre-eclampsia pregnancies.
EP 185 034 discloses one dimeric form of inhibin, which it identifies in terms of molecular weight, subunit structure and other properties. The sizes given are 14 kDxc2x12 kD and 44 kD. Later research suggests that the primary active form of inhibin A in biological fluids is a 32 kD molecule formed by a 12 kD xcex2A and 20 kD subunit. The 32 kD inhibin is the mature form produced by post-translational processing of precursor forms of molecular weight 65 kD and 56 kD. Immunoreactive xcex1 monomer also circulates in the body. The various different circulating inhibin proteins are often together referred to as xe2x80x9cinhibin formsxe2x80x9d.
It has also been discovered that maternal peripheral serum concentrations of the related hormones pro alpha C and activin A, when measured as total activin A, are significantly elevated in pre-eclampsia compared to normal maternal serum. Activins are homodimers consisting of xcex2Axcex2A (activin A), xcex2Axcex2B (activin AB) and xcex2Bxcex2B (activin B) subunits linked by disulphide bridges. Activin A occurs naturally in free form and in bound form, bound to a protein called follistatin. xe2x80x9cTotalxe2x80x9d activin A refers to both activin A whether free or bound. Pro alpha C is a part of the xcex1 subunit of inhibin A which is not present in the biologically active dimer. Serum human chorionic gonadotrophin (hCG) concentrations are also significantly higher in pre-eclampsia compared to normal pregnancy serum.
Khalil et al 1995 and Petraglia et al 1995 discuss hormone levels during normal and abnormal pregnancies but do not describe or suggest any possibility of a test for pre-eclampsia.
The present invention provides in one aspect a method of diagnosis of pre-eclampsia which method comprises measuring inhibin A in a biological sample.
Diagnosis according to the invention includes predictive diagnosis of pre-eclampsia, that is a prediction that the secondary symptoms of pre-eclampsia such as high blood pressure will occur.
Preferably, the sample is a maternal body fluid. The sample may be a serum or plasma sample from the maternal blood. Alternatively, it may be for example an amniotic fluid sample.
In another aspect, the invention provides the use of inhibin A levels as an indicator of pre-eclampsia.
The invention may further comprise measuring the level of other proteins, which may be hormones and the use of other such proteins together with inhibin A as indicators of pre-eclampsia. The additional proteins measured may be one or more of the hormones activin A, pro alpha C and hCG. Where activin A is measured, this is preferably total activin A.
The way in which measurement of inhibin A is carried out is not material to the invention. Recently developed specific and sensitive assays for inhibin A are described by Groome et al 1994; and Muttukrishna et al 1994. The presently preferred manner for measuring inhibin A in a biological sample uses one antibody specific for the alpha-subunit of inhibin A and a second antibody specific for the beta-subunit of inhibin A.
In yet another aspect, the invention provides the use of an antibody system specific for inhibin A in a test for pre-eclampsia.
In accordance with the invention, levels of all molecular forms of dimeric inhibin A are preferably measured, irrespective of molecular size. However, the measurement of specifically the mature 32 kD form of the protein may be sufficient for the purposes of the invention.
In accordance with the present invention, it has been demonstrated that inhibin A levels are significantly increased in pre-eclamptic pregnancies over normal pregnancies. Not only that, it has also been found that there is no overlap in inhibin A levels between pre-eclamptic and normal pregnancies. Furthermore, it has been demonstrated that at an early stage in gestation, before the secondary symptoms of pre-eclampsia are normally detectable, a significantly higher inhibin A level is detectable in a group of patients who go on to develop pre-eclampsia compared to a control group of individuals who do not have a pre-eclamptic pregnancy.
A clinically useful screening test for women at risk of pre-eclampsia has not previously existed. Detection of patients at high risk of pre-eclampsia before the disease develops will allow increased surveillance, monitoring of foetal growth and well-being and placental function. It may also lead to new methods of intervention to stabilise blood pressure and renal function and possibly reduce the severity of the disease. Any intervention which can prolong pregnancy safely will reduce the burden of prematurity which remains one of the major causes of foetal mortality and neonatal and childhood handicap.